Aberrant cell-restricted immunoglobulins provided with a toxic moiety

ABSTRACT

Described are immunoglobulins provided with a toxic moiety, comprising at least an immunoglobulin variable region that specifically binds to an MHC-peptide complex preferentially associated with aberrant cells. These immunoglobulins provided with a toxic moiety are preferably used in selectively modulating biological processes. The provided immunoglobulins provided with a toxic moiety are of particular use in pharmaceutical compositions for the treatment of diseases related to cellular aberrancies, such as cancers and autoimmune diseases.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) to U.S. Ser.No. 61/586,568, filed on Jan. 12, 2012, the contents of which areincorporated herein by this reference.

TECHNICAL FIELD

The disclosure relates to the field of biotechnology andbiotherapeutics. More specifically, it relates to immunoglobulinsprovided with a toxic moiety and human antibodies. It also relates tothe use of these biotherapeutics in the treatment of a host sufferingfrom a disease associated with aberrant cells, such as cancers andautoimmune diseases.

BACKGROUND

The development of immunoglobulin-drug conjugates is a drug developmentfield that receives great attention nowadays. Humanized or humanantibodies are the largest and most important class of immunoglobulinsunder investigation for use in antibody-drug conjugates (ADCs) and inimmunotoxins and antibody-radionuclide conjugates. These antibodiestarget binding sites (over)expressed at aberrant cells, such as thoseexposed in cancers and immune or autoimmune diseases, and duringinfections. Many of the conjugates have a limited degree of efficacy.For example, the maximum tolerated dose of immunotoxins is relativelylow due to their toxicity towards healthy tissue. Lowering the dose isone way of protecting healthy cells for the non-specific toxic activityof the toxin or the drug in ADCs. Lowering the dose, however, hampersthe delivery of an efficacious amount of conjugate at the site of forexample a tumor. The unwanted side reactions are mainly due to thetargeting of the antibodies to binding sites that are not exclusivelyexposed by aberrant cells but also to some extent by healthy cells.Thus, insufficient specificity for aberrant cells over healthy cellshampers desired efficacy and hampers obtaining the desired safetyprofiles of the nowadays immunoglobulin-drug conjugates.

Toxic moieties currently in the clinic or under investigation arenumerous and diverse.^([6]) Amongst the first toxins that werechemically linked to murine antibodies are plant-derived protein toxinsand bacterial toxins such as saporin, Diphtheria toxin, Pseudomonasexotoxin, gelonin, ricin, ricin A chain, abrin and pokeweed antiviralprotein. Other immunoglobulins provided with a toxin moiety comprisesingle chain Fv fused at the DNA level with toxins. An example is therecombinant protein BL22 consisting of the Fv portion of an anti-humanCD22 antibody fused to a fragment of Pseudomonas exotoxin-A, thattargets B-cell malignancies such as hairy cell leukemia andnon-Hodgkin's lymphoma. Other examples of immunoglobulins conjugated totoxins are the antibody-radionuclide conjugates. Human CD20 has beenchosen by drug developers as the target for two monoclonal antibodies,conjugated with 90-Yttrium or with 131-Iodine, for treatment ofnon-Hodgkin's lymphomas. In attempts to improve the tumor selectivity ofcertain drugs, murine monoclonal antibodies were conjugated to compoundssuch as doxorubicin, vinblastine, methotrexate, providing so-calledantibody-drug conjugates. Insufficient tumor cell specificity howeverstill limited the therapeutic usefulness. Even when selecting tumor cellsurface antigens that are (highly) over-expressed at aberrant cells,still the low expression levels at healthy cells gives rise toinsufficient selectivity of the antibody-drug conjugates. Currentcytotoxic anti-tumor drugs under investigation are for examplemaytansinoids and dolastatin analogs, that both target intracellulartubulin, and duocarmycins and calicheamicins, which target DNAstructure. These compounds are potent in their cytotoxic activity,though not selective for aberrant cells. Antibiotic calicheamicinconjugated to an anti-human CD33 monoclonal antibody was approved andused in the clinic, but was withdrawn due to serious side effects.Additional examples of drugs currently under investigation for theirpotential beneficial use in antibody-drug conjugates meant for thetreatment of cellular aberrancies are ozogamicin,hydrazone-calicheamicin, vedotin, emtansine, mertansine. These toxicmoieties are conjugated to immunoglobulins targeting cell surfacemarkers expressed at tumor cells, though also expressed to some extentat healthy cells. Typical examples of immunoglobulin-drugconjugate-targeted cell surface markers present at both tumor cells andhealthy cells are CD19, CD20, CD22, CD25, CD30, CD33, CD56, CD70,HER2/neu. All these immunoglobulin-drug conjugate development programsthus inherently bear the risk for unacceptable safety profiles andconsequent poor efficacy due to low maximum tolerated doses. Conjugatingdrugs, radionuclides or toxins to immunoglobulins specifically andselectively targeting aberrant cells and not targeting healthy cellswould thus provide for therapies with improved specificity andselectivity for aberrant cells and with an improved safety profile.

SUMMARY OF THE DISCLOSURE

Specific and selective delivery of a toxic moiety in target aberrantcells demands for binding molecules specific for binding sitespreferentially associated with aberrant cells. These binding moleculesthen are used as carriers and transporters of the toxic moieties,specifically and selectively delivering the toxic moieties at and in theaberrant cells. Herein, we disclose immunoglobulin-drug conjugatescomprising these preferred features. The immunoglobulins in theimmunoglobulin-drug conjugates hereof comprise immunoglobulin bindingregions with improved selectivity for aberrant cells by specificallybinding to binding sites preferentially associated with these aberrantcells. We disclose as preferred targets for the antibody hereof,intracellular proteins that are associated with aberrant cells. Theseproteins are available as peptides presented by MHC on the surface ofaberrant cells. The use of MHC-peptide complexes as targets opens us anew field of tumor targets, because so far typically targets associatedwith the surface of aberrant cells have been envisaged. Although it ispreferred that the target is specific for aberrant cells (tumor cells)in many cases up-regulated intracellular proteins are also suitable forat least improving the therapeutic window of immunotoxins. Our mostpreferred targets are peptides derived from MAGE presented in thecontext of MHC-1. In particular MAGE peptides that are present in morethan one MAGE protein (multi-MAGE epitope; see WO2012/091564incorporated herein by reference). The toxic moiety for use herein ispreferably a drug compound, a radionuclide or a toxin. A toxic moiety isa non-proteinaceous molecule or a proteinaceous molecule. In theimmunoglobulin-drug conjugates hereof, the toxic moiety is preferablyconjugated by chemical conjugation. Also preferred are immunoglobulinshereof fused at the DNA level to a proteinaceous toxic moiety.

The immunoglobulins in the immunoglobulin-drug conjugates hereof aresuitable for the specific and selective localization of a toxic effectinside targeted aberrant cells, leaving healthy cells essentiallyunaffected. Immunoglobulins comprise immunoglobulin binding domains,referred to as immunoglobulin variable domains, comprisingimmunoglobulin variable regions. Maturation of immunoglobulin variableregions results in variable domains adapted for specific binding to atarget binding site. Immunoglobulins are therefore particularly suitablefor providing the immunoglobulin-drug conjugates hereof with the abilityto specifically and selectively target aberrant cells. At their surface,aberrant cells present aberrant cell-associated antigen peptides in thecontext of major histocompatibility complex (MHC). Therefore, for theimmunoglobulins in the immunoglobulin-drug conjugates hereof, aberrantcell-associated MHC-1 peptide complexes are a preferred target onaberrant cells. In addition, aberrant cell-associated MHC-2 peptidecomplexes are valuable targets on, e.g., tumors of hematopoietic origin,for the immunoglobulins in the immunoglobulin-drug conjugates hereof.Therefore provided are immunoglobulins in immunoglobulin-drugconjugates, with improved specificity and selectivity for aberrant cellsby targeting MHC-peptide complexes which are preferentially associatedwith aberrant cells. This improved specificity and selectivity foraberrant cells is accompanied with a reduced level of unintentionaltargeting of healthy cells by the immunoglobulins in theimmunoglobulin-drug conjugates hereof. Most preferably, healthy cellsare not targeted by the immunoglobulin-drug conjugates hereof.

Thus, in a first embodiment the invention provides an immunoglobulinprovided with a toxic moiety, comprising at least an immunoglobulinvariable region that specifically binds to an MHC-peptide complexpreferentially associated with aberrant cells. Preferred immunoglobulinshereof are antibodies, but fragments and/or derivatives such as Faband/or ScFv can also be used. Even more preferred immunoglobulins hereofare antibodies of the immunoglobulin G (IgG) type. Other immunoglobulinshereof are for example heavy-chain (only) antibodies comprising Vh orVhh and IgA, and their fragments such as Fab fragments, and Fabfragments of IgGs. Immunoglobulins bind via their immunoglobulinvariable regions to binding sites on molecules, such as epitopes, with ahigher binding affinity than background interactions between molecules.In the context hereof, background interactions are typicallyinteractions with an affinity lower than a K_(D) of 10E-4 M.Immunoglobulin variable domains in light chains (VI) and immunoglobulinvariable domains in heavy chains (Vh) of antibodies typically comprisethe aberrant cell-specific immunoglobulin variable regions hereof.

Thus, in one embodiment, provided is an immunoglobulin provided with atoxic moiety, comprising at least an immunoglobulin variable region,wherein the immunoglobulin variable region is a Vh(h) that specificallybinds to an MHC-peptide complex preferentially associated with aberrantcells. Thus, in yet another embodiment, also provided is animmunoglobulin provided with a toxic moiety, comprising at least animmunoglobulin variable region, wherein the immunoglobulin variableregion is a Vh that specifically binds to an MHC-peptide complexpreferentially associated with aberrant cells, and wherein theimmunoglobulin variable region further comprises a Vl.

As said, immunoglobulins G are particularly suitable binding moleculesfor use in therapies specifically and selectively targeting aberrantcells, for site-specific delivery of a toxic moiety hereof. Because theanticipated predominant use of the antibodies hereof is in therapeutictreatment regimes meant for the human body, in a particular embodimenthereof, the immunoglobulins provided with a toxic moiety have anamino-acid sequence of human origin. Thus, in one embodiment, providedis a human IgG provided with a toxic moiety, comprising at least animmunoglobulin variable region, wherein the immunoglobulin variableregion is a Vh that specifically binds to an MHC-peptide complexpreferentially associated with aberrant cells, and wherein theimmunoglobulin variable region further comprises a Vl. Of course,humanized antibodies, with the precursor antibodies encompassing aminoacid sequences originating from other species than human, are also parthereof. Also part hereof are chimeric antibodies, comprising (parts of)an immunoglobulin variable region hereof originating from a speciesother than human, and grafted onto a human antibody.

An aberrant cell is defined as a cell that deviates from its healthynormal counterparts. Aberrant cells are for example tumor cells, cellsinvaded by a pathogen such as a virus, and autoimmune cells.

Thus, in one embodiment, provided is an immunoglobulin according to anyof the aforementioned embodiments wherein the MHC-peptide complex isspecific for aberrant cells.

In the molecules hereof, the toxic moieties are preferably chemicallylinked to the immunoglobulins via any linker chemistry know in the art,and optionally via an additional spacer. Hereof, one or several,preferably two to six toxic moiety molecules are chemically linked to animmunoglobulin molecule hereof. The number of conjugated toxic moietymolecules per single immunoglobulin molecule is restricted by boundariessuch as the number of available sites for conjugation on theimmunoglobulin, the stability of the conjugate, the preservation of theability of the immunoglobulin to specifically bind to an aberrant cell,etc. Of course, also two, three, etc., different toxic moieties can belinked to an immunoglobulin, depending amongst others on availablebinding sites and the applied linker chemistry. Chemical linking of thetoxic moieties has several advantages when working with immunoglobulins.This way, toxic moieties cannot interfere with expression, folding,assembly and secretion of the immunoglobulin molecules.

Thus, in one embodiment, provided is an immunoglobulin according to anyof the aforementioned embodiments wherein the toxic moiety is chemicallylinked to the immunoglobulin. It is then also part of the currentinvention that toxic moieties are covalently bound via peptide bonds,and preferably via a peptide linker, to the immunoglobulins hereof Thetoxic moiety and the immunoglobulin are then fused at the DNA level.

Thus, in one embodiment, provided is an immunoglobulin according to anyof the aforementioned embodiments wherein the toxic moiety is a protein,preferably fused to the immunoglobulin at the DNA level, preferablythrough a linker sequence. In many instances, a simple Gly-Ser linker of4-15 amino-acid residues may suffice, but if greater flexibility betweenthe immunoglobulin and the toxic moiety is desired, longer or morecomplex linkers may be used. Preferred linkers are (Gly₄Ser)_(n),(GlySerThrSerGlySer)_(n), GlySerThrSerGlySerGlyLysProGlySerGlyGluGlySerThrLysGly,GlyPheAlaLysThrThrAlaProSerValTyrProLeuAlaProVal LeuGluSerSerGlySerGly(SEQ ID NO:105) or any other linker that provides flexibility allowingprotein folding, stability against undesired proteolytic activity andflexibility for the immunoglobulins hereof to exert their activity.

Another group of preferred linkers are linkers based on hinge regions ofimmunoglobulins. These linkers tend to be quite flexible and quiteresistant to proteases. The most preferred linkers based on hingeregions are GluProLysSerCysAspLysThrHisThr (linking Ch1 and Ch2 in IgG1)(SEQ ID NO:106), GluLeuLysThrProLeuGlyAspThrThrHisThr (IgG3) (SEQ IDNO:107), and GluSerLysTyrGlyProPro (IgG4) (SEQ ID NO:108). Thus, therole of any applied chemical linker in conjugates hereof or the role ofany applied peptide linker in fused molecules hereof is aiding the dualactivity of the antibodies hereof, i.e., specific and selective bindingof the immunoglobulin to aberrant cells, and subsequent delivery of atleast the toxic moiety in the targeted aberrant cells. Thus, in oneembodiment, provided is the use of an immunoglobulin provided with atoxic moiety according to any of the aforementioned embodiments, for thetreatment of a host suffering from a disease associated with aberrantcells. In a further embodiment, provided is the use of an immunoglobulinprovided with a toxic moiety according to any of the aforementionedembodiments, for the treatment of a host suffering from a diseaseassociated with aberrant cells wherein at least the toxic moiety isinternalized into the aberrant cell. The immunoglobulins provided with atoxic moiety are for example used for the treatment of cancer. Thus, inone embodiment, provided is an immunoglobulin provided with a toxicmoiety according to any of the aforementioned embodiments for use in thetreatment of cancer.

Preferred toxic moieties are numerous. Several examples of preferredtoxic moieties hereof are drugs such as doxorubicin, cisplatin,carboplatin, vinblastine, methotrexate, chelated radioactive metal ions,(synthetic) antineoplastic agents such as monomethyl auristatin E,radioactive iodine, radionuclides such as 90-Yttrium, 131-Iodine, toname a few, which are chemically conjugated to the immunoglobulinshereof. Also preferred toxic moieties are proteinaceous toxins such as afragment of Pseudomonas exotoxin-A, statins, ricin A, gelonin, saporin,interleukin-2, interleukin-12, viral proteins E4orf4, apoptin and NS1,and non-viral proteins HAMLET, TRAIL and mda-7. Thus, in one embodimenthereof, antibodies are provided for the specific targeting of aberrantcells, wherein the toxic moiety is selected from the list of availabletoxic moieties comprising toxins such as a fragment of Pseudomonasexotoxin-A, statins, chelated radioactive metal ions, radioactiveiodine, ricin A, gelonin, saporin, interleukin-2, interleukin-12,radionuclides such as 90-Yttrium, 131-Iodine, drugs such as doxorubicin,taxol or derivatives, 5-FU, anthracyclines, vinca alkaloids,calicheamicins, cisplatin, carboplatin, vinblastine, methotrexate,(synthetic) antineoplastic agents such as monomethyl auristatin E,apoptin, parvovirus-H1 NS1 protein, E4orf4, TRAIL, mda-7, HAMLET.

Proteinaceous molecules are molecules comprising at least a string ofamino acid residues. In addition, hereof the proteinaceous molecules maycomprise carbohydrates, disulphide bonds, phosphorylations,sulphatations, etc.

When antibodies hereof are designed to first bind to a target aberrantcell, followed by internalization, the toxic moiety can thensubsequently have its intracellular (cytotoxic) function, i.e., inducingapoptosis.

For administration to subjects the antibodies hereof must be formulated.Typically these antibodies will be given parenterally. For formulationsimply water (saline) for injection may suffice. For stability reasonsmore complex formulations may be necessary. The invention contemplateslyophilized compositions as well as liquid compositions, provided withthe usual additives. Thus, in one embodiment, provided is apharmaceutical composition comprising an immunoglobulin provided with atoxic moiety according to any of the aforementioned embodiments andsuitable diluents and/or excipients.

The dosage of the antibodies hereof are established through animalstudies, (cell-based) in vitro studies, and clinical studies inso-called rising-dose experiments. Typically, the doses will becomparable with present day antibody dosages (at the molar level).Typically, such dosages are 3-15 mg/kg body weight, or 25-1000 mg perdose.

In addition, especially in the more difficult to treat cellularaberrancies the first applications of the antibodies hereof will (atleast initially) probably take place in combination with othertreatments (standard care). Of course, also provided are antibodies foruse in novel or first treatments of any malignancy accompanied by theoccurrence of aberrant cells, for which current treatments are notefficient enough or for which currently no treatment options areavailable. Thus, for example, also provided is a pharmaceuticalcomposition comprising an invented immunoglobulin provided with a toxicmoiety and a conventional cytostatic and/or tumoricidal agent. Moreover,also provided is a pharmaceutical composition comprising an inventedimmunoglobulin provided with a toxic moiety for use in an adjuvanttreatment of cancer. Thus, in one embodiment hereof, an inventedimmunoglobulin provided with a toxic moiety for use in an adjuvanttreatment of cancer is provided. Additionally, also provided is apharmaceutical composition comprising an invented immunoglobulinprovided with a toxic moiety for use in a combination chemotherapytreatment of cancer. Examples of chemotherapeutical treatments that arecombined with the pharmaceutical composition of the current inventionare etoposide, paclitaxel, cisplatin, doxorubicin and methotrexate.

The pharmaceutical compositions hereof will typically find their use inthe treatment of cancer, particularly in forms of cancer where thetargets of the preferred antibodies hereof (complexes of MHC andtumor-specific antigen peptides) are presented by the tumors. Table 1,for example, gives a list of tumors on which complexes of MHC and MAGE-Apeptides have been found. It is easy using an antibody hereof toidentify tumors that present these target MHC-peptide complexes. Thiscan be done in vitro or in vivo (imaging).

It is preferred that the cell-surface molecules comprising the bindingsites for the antibodies hereof are internalized into the targetedaberrant cell, together with the antibodies hereof, or together with atleast the toxic moiety of the antibodies hereof. In a particularlypreferred embodiment hereof the targeted aberrant cells go intoapoptosis as a result of the internalization. Thus, in one embodiment,provided is the use of an immunoglobulin provided with a toxic moietyaccording to any of the aforementioned embodiments, for the treatment ofa host suffering from cancer, wherein at least the toxic moiety isinternalized into the aberrant cell.

Also comprised herein is a nucleic acid molecule encoding theimmunoglobulin part of an antibody according to any of the embodimentshereof, when the toxic moiety is chemically linked to the immunoglobulinin the antibody hereof. Thus, also comprised herein is a nucleic acidmolecule encoding an immunoglobulin and a toxic moiety according to anyof the embodiments hereof, when the toxic moiety is fused to theimmunoglobulin at the DNA level. These molecules hereof can be producedin prokaryotes or eukaryotes. The codon usage of prokaryotes may bedifferent from that in eukaryotes. The nucleic acids hereof can beadapted in these respects. Also, elements that are necessary forsecretion may be added, as well as promoters, terminators, enhancers,etc. Also, elements that are necessary and/or beneficial for theisolation and/or purification of the immunoglobulins hereof or of theantibodies hereof may be added. Typically, the nucleic acids hereof areprovided in an expression vector suitable for the host in which they areto be produced. Choice of a production platform will depend on the sizeof the molecule, the expected issues around protein folding, whetheramino acid sequences are present in the immunoglobulin or in theantibody that require glycosylation, expected issues around isolationand/or purification, etc. For example, the presence of disulfide bondsin immunoglobulins or proteinaceous toxins hereof will typically guidethe selection of the preferred production platform. Thus, typicallynucleic acids hereof are adapted to the production and purificationplatform in which the immunoglobulins optionally with their fusedproteinaceous toxins hereof are to be produced. Thus, provided is avector comprising a nucleic acid molecule encoding an immunoglobulin oran antibody hereof. For stable expression in a eukaryote it is preferredthat the nucleic acid encoding the immunoglobulin or the antibody hereofis integrated in the host cell genome (at a suitable site that is notsilenced). In one embodiment, also provided is a vector comprising meansfor integrating the nucleic acid in the genome of a host cell. Thedisclosure further comprises the host cell or the organism in which thenucleic acid molecule encoding for the immunoglobulin hereof optionallywith their fused proteinaceous toxins, is present and which is thuscapable of producing the immunoglobulin optionally with their fusedproteinaceous toxins hereof. Thus, in a preferred embodiment, alsoprovided is a cell comprising a nucleic acid molecule hereof, preferablyintegrated in its genome and/or a vector hereof, comprising a nucleicacid molecule encoding an immunoglobulin optionally with their fusedproteinaceous toxins hereof.

Included herein invention is also a method for producing animmunoglobulin optionally with their fused proteinaceous toxins hereof,comprising culturing a cell hereof, comprising a nucleic acid moleculeencoding an immunoglobulin optionally with their fused proteinaceoustoxins hereof, preferably integrated in the cell's genome and/or avector hereof, comprising a nucleic acid molecule encoding animmunoglobulin optionally with their fused proteinaceous toxins hereof,allowing for expression of the immunoglobulin optionally with theirfused proteinaceous toxins and separating the immunoglobulin optionallywith their fused proteinaceous toxins from the culture.

In one embodiment hereof, the immunoglobulin variable domains in themolecules hereof target one binding site. Also bi-specificimmunoglobulins provided with a toxic moiety are provided that arespecifically binding to two different binding sites associated with thecell surface of aberrant cells. By targeting with a single antibodyhereof two different binding sites on an aberrant cell such as a tumorcell, the risk that both targets are also jointly present on a healthycell is significantly further diminished. The affinity of the antibodieshereof for the two different target binding sites separately, preferablyis designed such that K_(on) and K_(off) are very much skewed towardsbinding to both different binding sites simultaneously. Thus, thespecificity of the bi-specific antibodies hereof is increased byincreasing their specificity for binding to two different binding sitesassociated with aberrant cells. Thus, in one embodiment hereof, theantibody according to any of the previous embodiments is ahetero-dimeric bi-specific immunoglobulin G or heavy-chain only antibodycomprising two different but complementary heavy chains. The twodifferent but complementary heavy chains may then be dimerized throughtheir respective Fc regions. Upon applying preferred pairingbiochemistry, hetero-dimers are preferentially formed over homo-dimers.For example, two different but complementary heavy chains are subject toforced pairing upon applying the “knobs-into-holes” CH3 domainengineering technology as described (Ridgway et al., ProteinEngineering, 1996 (ref 14)). In a preferred embodiment hereof the twodifferent immunoglobulin variable regions in the bi-specificimmunoglobulins hereof specifically bind to an MHC-peptide complexpreferentially associated with aberrant cells.

Typical preferred antibodies hereof are exemplified by the antibodiesoutlined in this section, in FIG. 5B, and by the examples provided belowand in the Examples section. Thus the invention provides animmunoglobulin provided with a toxic moiety according to FIG. 5B.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Specific binding of HLA-A0201/multi-MAGE-A-specific phage clonesisolated from a large human non-immune antibody Fab phage library.Individual antibody Fab expressing phages that were selected againstbiotinylated HLA-A0201/multi-MAGE-A were analyzed by ELISA for theircapacity to bind the relevant peptide/MHC complex only. Streptavidincoated 96-well plates were incubated with soluble HLA-A0201/multi-MAGE-A(A2/multiMage) or HLA-A0201/JCV (A2/JC) peptide/MHC complexes (10μg/ml), washed to remove non-bound complexes and incubated withindividual phage clones. Non-binding phages were first removed by threewashes with PBS/TWEEN®, followed by incubation with anti-M13 antibody (1μg/ml, Amersham) for one hour by room temperature. Finally the wellswere incubated with an HRP-labeled secondary antibody and bound phagesdetected.

FIG. 2: Phages AH5, CB1 and CG1 specifically bind cells presenting themulti-MAGE-A peptide. Phages AH5, CB1, CG1, BD5 and BC7 that had shownspecific binding in ELISA using the relevant HLA-A201/multi-MAGE-Acomplex and an irrelevant HLA-A201 complex loaded with a JCV peptidewere analyzed for their capacity to bind cells presenting themulti-MAGE-A peptide in HLA-A0201 molecules. To this end, human B-LCL(BSM) were loaded with multi-MAGE-A peptide (10 μg in 100 μl PBS) for 30minutes at 37° C., followed by incubation with the Fab phages AH5, CB1,CG1, BD5 and BC7 and analyzed by flow-cytometry using anti-phageantibodies and a fluorescently labeled secondary antibody.

FIG. 3: Phages expressing HLA-A2/multi-MAGE-A-specific Fab bind tumorcells of distinct histologic origin. Phages AH5, CB1 and CG1 specificfor HLA-A0201/multi-MAGE-A and a positive control phage specific forHA-0101/MAGE-A1 were used for staining of distinct tumor cell lines. Tothis end the prostate cancer cell line LNCaP, the multiple myeloma cellline MDN, the melanoma cell lines MZ2-MEL43 and G43, and the breastcancer cell line MDA-MD157 were incubated with the different phages (30minutes at 4° C.), bound phages were then detected by flow cytometryusing anti-phage antibodies and fluorescently labeled secondaryantibodies.

FIG. 4: Phage AH5 specifically binds HLA-A0201/multi-MAGE-A complexesonly. To determine specificity of the phage AH5 an ELISA was performedusing relevant and irrelevant peptide/MHC complexes. HLA-A0201 withmulti-MAGE-A, gp100, JCV and MAGE-C2 peptides, as well as HLA-A1 withMAGE-A1 peptide were coated on streptavidin 96-well plates and incubatedwith phage AH5.

FIG. 5: Cartoon displaying examples of preferred immunoglobulinsprovided with a toxic moiety, hereof.

Panel A: Cartoon displaying the topology of the twelve immunoglobulindomains assembled in an immunoglobulin G.

Panel B: Examples are provided of preferred immunoglobulins providedwith a toxic moiety, hereof Shown are immunoglobulins provided with asingle toxic moiety such as for example a cytostatic agent, linked tothe immunoglobulin with a chemical linker (exemplified by I. and II.;immunoglobulin-toxic moiety conjugates), or immunoglobulins providedwith a single toxic moiety, linked to the immunoglobulin with a peptidelinker (exemplified by III.; fused immunoglobulin-toxic moietymolecule). In IV., an immunoglobulin provided with a toxic moiety,hereof, is shown, comprising one immunoglobulin heavy chain comprising afused proteinaceous toxic moiety, comprising immunoglobulin variableregions specific for a certain binding site, and comprising a secondimmunoglobulin heavy chain comprising immunoglobulin variable regionsspecific for a different binding site. Of course, also part hereof arebi-specific immunoglobulins provided with a toxic moiety, hereof,comprising two heavy chains comprising different immunoglobulin variableregions specific for different binding sites and further comprising thesame or different proteinaceous toxic moieties fused two the heavychains. Of course, as part hereof, more than one, and typically two tosix toxic moiety molecules can be fused or conjugated to animmunoglobulin molecule.

FIG. 6: Human Fab phage F9 specifically binds HLA-A2/FLWGPRALV (SEQ IDNO:23) positive CMT64 mouse lung tumor cells. Human Fab clone F9 wasanalyzed for its capacity to bind mouse lung tumor cells (CMT64) stablyexpressing the HLA-A2/FLWGPRALV (SEQ ID NO:23) complex. Purified CloneF9 Fab fragments (3 μg total) were incubated with 0.5×10⁶ CMT64 cellsthat do not express human HLA, that express HLA-A2/YLEYRQVPG (SEQ IDNO:3) or that express HLA-A2/FLWGPRALV (SEQ ID NO:23). After one hourincubation on ice CMT64 cells were incubated with a fluorescentlylabeled secondary antibody and analyzed by flow cytometry.

FIG. 7: Llama VHH specifically binds CMT64 mouse lung tumor cellsexpressing human HLA-A2/multi-MAGE-A. Llama VHH specific for A2/FLW orA2/YLE were analyzed by flow cytometry for their binding capacity toCMT64 cells expressing these human HLA-A0201/multi-MAGE-A complexes.Purified VHH fragments (3 μg total) were incubated with 0.5×10⁶ CMT64cells, which do not express human HLA, that express HLA-A2/YLEYRQVPG(SEQ ID NO:3) or that express HLA-A2/FLWGPRALV (SEQ ID NO:23). After onehour incubation on ice CMT64 cells were incubated with a fluorescentlylabeled secondary antibody and analyzed by flow cytometry.

DETAILED DESCRIPTION

One aspect hereof relates to a method for providing the antibodieshereof. As described hereinabove, it typically involves providing anucleic acid construct encoding the desired immunoglobulin part ofantibodies hereof, or encoding the desired immunoglobulin fused to aproteinaceous toxic moiety. The nucleic acid construct can beintroduced, preferably via a plasmid or expression vector, into aprokaryotic host cell and/or in a plant cell and/or in a eukaryotic hostcell capable of expressing the construct. In one embodiment, a methodhereof to provide an immunoglobulin or to provide an immunoglobulinfused to a proteinaceous toxic moiety comprises the steps of providing ahost cell with the nucleic acid(s) encoding the immunoglobulin or theimmunoglobulin fused to a proteinaceous toxic moiety, and allowing theexpression of the nucleic acid(s) by the host cell.

It is part hereof that nucleic acids coding for selected (human)immunoglobulin Vh(h) domains according to any of the above embodimentsare combined with nucleic acids coding for human immunoglobulin heavychain constant domains, providing nucleic acid molecules hereof encodingfor a heavy chain of a human antibody. The human antibody heavy chainprotein product of such a nucleic acid molecule hereof, then may behetero-dimerized with a universal human antibody light chain. It is alsopart hereof that nucleic acids coding for (jointly) selected humanimmunoglobulin Vl domains and Vh domains according to any of the aboveembodiments are combined with nucleic acids coding for a humanimmunoglobulin light chain constant domain and are combined with nucleicacids coding for human immunoglobulin heavy chain constant domains,respectively, providing nucleic acid molecules hereof encoding for alight chain and for a heavy chain of a human antibody. In yet anotherembodiment hereof, the nucleic acids coding for the complementaritydetermining regions 1, 2 and 3 (CDR1, CDR2, CDR3), forming together theimmunoglobulin variable region of a selected immunoglobulin Vh domainand/or a selected immunoglobulin Vl domain according to any of the aboveembodiments are combined with nucleic acids coding for humanimmunoglobulin Vh domain frame work regions and/or human immunoglobulinVl domain frame work regions, respectively, providing nucleic acidmolecules hereof encoding for a heavy chain variable domain (Vh) of ahuman antibody and/or encoding for a light chain variable domain (Vl) ofa human antibody (A method known in the art as “grafting”). Thesenucleic acid molecules encoding for variable domains Vh and/or Vl are,as part hereof, then combined with nucleic acids coding for humanimmunoglobulin constant domains, providing a nucleic acid moleculeencoding for a human antibody heavy chain and/or providing a nucleicacid molecule encoding for a human antibody light chain.

Hereof, immunoglobulins or immunoglobulins fused to a proteinaceoustoxic moiety are for example expressed in plant cells, eukaryotic cellsor in prokaryotic cells. Non-limited examples of suitable expressionsystems are tobacco plants, Pichia pastoris, Saccharomyces cerevisiae.Also cell-free recombinant protein production platforms are suitable.Preferred host cells are bacteria, like for example bacterial strainBL21 or strain SE1, or mammalian host cells, more preferably human hostcells. Suitable mammalian host cells include human embryonic kidney(HEK-293) cells, PERC6® cells or preferably Chinese hamster ovary (CHO)cells, which can be commercially obtained. Insect cells, such as S2 orS9 cells, may also be used using baculovirus or insect cell expressionvectors, although they are less suitable when the immunoglobulins or thefused immunoglobulins-toxic moiety molecules hereof include elementsthat involve glycosylation. The produced immunoglobulins or fusedimmunoglobulin-toxic moiety molecules hereof can be extracted orisolated from the host cell or, if they are secreted, from the culturemedium of the host cell. Thus, in one embodiment a method hereofcomprises providing a host cell with one or more nucleic acid(s)encoding the immunoglobulin or the fused immunoglobulin-toxic moietymolecule, allowing the expression of the nucleic acids by the host cell.In another preferred embodiment a method hereof comprises providing ahost cell with one or more nucleic acid(s) encoding two or moredifferent immunoglobulins or two or more different fusedimmunoglobulin-toxic moiety molecules, allowing the expression of thenucleic acids by the host cell. For example, in one embodiment, nucleicacids encoding for a so-called universal immunoglobulin light chain andnucleic acids encoding for two or more different immunoglobulin heavychains are provided, enabling isolation of mono-specific immunoglobulinsor mono-specific fused immunoglobulin-toxic moiety molecules comprisinghomo-dimers of heavy chains and/or enabling isolation of bi-specificimmunoglobulins or bi-specific fused immunoglobulin-toxic moietymolecules comprising hetero-dimers of heavy chains, with all differentheavy chains complexed with a universal light chain. Methods for therecombinant expression of (mammalian) proteins in a (mammalian) hostcell are well known in the art.

As said, it is preferred that the immunoglobulins hereof are linked withthe toxic moieties via bonds and/or binding interactions other thanpeptide bonds. Methods for linking proteinaceous molecules such asimmunoglobulins to other proteinaceous molecules or non-proteinaceousmolecules are numerous and well known to those skilled in the art ofprotein linkage chemistry. Protein linkage chemistry not based onpeptide bonds can be based on covalent interactions and/or onnon-covalent interactions. A typical example of linkage chemistriesapplicable for linking toxic moieties to immunoglobulins hereof are thevarious applications of the Universal Linkage System disclosed in patentapplications WO92/01699, WO96/35696, WO98/45304, WO03040722, thecontents of each of which are incorporated herein by this reference.

As will be clear, an antibody hereof finds its use in many therapeuticapplications and non-therapeutic applications, e.g., diagnostics, orscientific applications. Antibodies hereof, or more preferably theimmunoglobulin part of the antibodies hereof, suitable for diagnosticpurposes are of particular use for monitoring the expression levels ofmolecules exposing binding sites on aberrant cells that are targeted byantibodies hereof. In this way, it is monitored whether the therapyremains efficacious or whether other antibodies hereof targeting one ortwo different binding sites on the aberrant cells should be appliedinstead. This is beneficial when the expression levels of the first orthe first two targeted binding site(s) are below a certain threshold,whereas another or new binding sites (still) can serve as newly targetedbinding sites for antibodies hereof comprising the appropriate specificimmunoglobulin variable regions for these alternative binding site(s).Antibodies hereof may also be used for the detection of (circulating)tumor cells, and for the target-cell specific delivery ofimmune-stimulatory molecules. For these later two uses, the soleimmunoglobulins hereof without the fused or conjugated toxic moiety mayalso be used.

Provided herein is a method for inducing ex vivo or in vivo a modulatingeffect on a biological process in a target cell, comprising contactingthe cell with an antibody hereof in an amount that is effective toinduce the modulating effect. Preferably, the antibody hereof is usedfor a modulating effect on a biological process of aberrant cells in asubject, more preferably a human subject. For therapeutic applicationsin humans it is of course preferred that an antibody hereof does notcontain amino acid sequences of non-human origin. More preferred areantibodies hereof, which only contain human amino acid sequences.Therefore, a therapeutically effective amount of an antibody hereofcapable of recognizing and binding to one or two disease-specificbinding sites and subsequently inducing a modulating effect on abiological process in the cell, can be administered to a patient tostimulate eradication of aberrant cells expressing the binding site(s)without affecting the viability of (normal) cells not expressing thedisease-specific binding site(s). The specific killing of aberrant cellswhile minimizing or even avoiding the deterioration or even death ofhealthy cells will generally improve the therapeutic outcome of apatient after administration of the antibodies hereof.

Accordingly, also provided is the use of an antibody hereof asmedicament. In another aspect, provided is the use of an antibody hereoffor the manufacture of a medicament for the treatment of cancer,autoimmune disease, infection or any other disease of which the symptomsare reduced upon targeting aberrant cells expressing disease-specificbinding sites with antibodies hereof. For example, an antibody hereof isadvantageously used for the manufacture of a medicament for thetreatment of various cancers (e.g., solid tumors, hematologicmalignancies).

An example of a preferred antibody hereof is an antibody comprising atleast an immunoglobulin variable region specifically binding to thecomplex between MHC-1 HLA-0201 and a multi-MAGE-A epitope, conjugatedwith a toxic moiety, using for example Universal Linkage System linkerchemistry for conjugation. A second example of a preferred antibodyhereof is an antibody comprising at least an immunoglobulin variableregion specifically binding to the complex between MHC-1 HLA-CW7 and amulti-MAGE-A epitope, conjugated with a toxic moiety, using for exampleUniversal Linkage System linker chemistry for conjugation. With thebi-specific antibodies hereof, difficult to target and/or difficult toreach aberrant cells have a higher chance of being “hit” by at least oneof the two different immunoglobulin variable regions in the bi-specificantibodies hereof, thereby providing at least in part the therapeuticactivity. An example of a preferred bi-specific antibody hereof is animmunoglobulin comprising an immunoglobulin variable region specific forthe complex between MHC-1 HLA-0201 and a multi-MAGE-A epitope andcomprising a second immunoglobulin variable region specific for thecomplex between MHC-1 HLA-CW7 and a second multi-MAGE-A epitope,conjugated with a toxic moiety.

Antibody fragments of human origin can be isolated from large antibodyrepertoires displayed by phages. One aspect hereof, known by the art, isthe use of human antibody phage display libraries for the selection ofhuman antibody fragments specific for a selected binding site, e.g., anepitope. Examples of such libraries are phage libraries comprising humanVh repertoires, human Vh-Vl repertoires, human Vh-Ch1 or human antibodyFab fragment repertoires.

Although the disclosure contemplates many different combinations of MHCand antigenic peptides the most preferred is the combination of MHC-1and an antigenic peptide from a tumor related antigen presented by theMHC-1, exclusively expressed by aberrant cells and not by healthy cells.Because of HLA restrictions, there are many combinations ofMHC-1-peptide complexes as well as of MHC-2-peptide complexes that canbe designed based on the rules for presentation of peptides in MHC.These rules include size limits on peptides that can be presented in thecontext of MHC, restriction sites that need to be present for processingof the antigen in the cell, anchor sites that need to be present on thepeptide to be presented, etc. The exact rules differ for the differentHLA classes and for the different MHC classes. We have found thatMAGE-derived peptides are very suitable for presentation in an MHCcontext. An MHC-1 presentable antigenic peptide with the sequenceY-L-E-Y-R-Q-V-P-G in MAGE-A (SEQ ID NO:3) was identified, that ispresent in almost every MAGE-A variant (multi MAGE peptide) and thatwill be presented by one of the most prevalent MHC-1 alleles in theCaucasian population (namely HLA-A0201). A second MAGE peptide that ispresented by another MHC-1 allele (namely HLA-CW7) and that is presentin many MAGE variants, like, for example, MAGE-A2, -A3, -A6 and -A12, isE-G-D-C-A-P-E-E-K (SEQ ID NO:4). These two combinations of MHC-1 andMAGE peptides together would cover 80% of the Caucasian population. Thesame approach can be followed for other MHC molecules, other HLArestrictions and other antigenic peptides derived from tumor-associatedantigens. Relevant is that the chosen antigenic peptide to elicit theresponse to must be presented in the context of an MHC molecule andrecognized in that context only. Furthermore, the antigenic peptide mustbe derived from a sufficiently tumor-specific antigen and the HLArestriction must occur in a relevant part of the population. One of theimportant advantages of the invention is that tumors that down regulatetheir targeted MHC-peptide complex, can be treated with a secondimmunoglobulin comprising at least one variable region binding to adifferent MHC-peptide complex based on the same antigen. If this one isdown regulated a third one will be available. For heterozygotes sixdifferent targets on MHC-1 may be available. Since cells need to be“inspected” by the immune system from time to time, escape through downregulation of all MHC molecules does not seem a viable escape route. Inthe case that MAGE is the antigen from which the peptide is derivedescape through down regulation of the antigen is also not possible,because MAGE seems important for survival of the tumor.^([8]) Thus theinvention, in an important aspect reduces or even prevents escape of thetumor from the therapy. Thus, provided is in a preferred embodiment anantibody hereof whereby the immunoglobulin variable region is capable ofbinding to an MHC-I-peptide complex. In a further preferred embodimentthe invention provides an immunoglobulin whereby the immunoglobulinvariable region is capable of binding to MHC-I-peptide complexescomprising an antigenic peptide derived from a tumor related antigen, inparticular MHC-I-peptide complexes comprising an antigenic peptidepresent in a variety of MAGE antigens, whereby the immunoglobulin isprovided with a toxic moiety.

Because in one embodiment the invention uses MHC molecules as a target,and individuals differ in the availability of MHC targets, the inventionalso provides a so-called companion diagnostic to determine the HLAcomposition of an individual. Although the invention preferably uses amore or less universal (MAGE) peptide, the invention also provides adiagnostic for determining the expression of the particular antigen bythe tumor. In this manner the therapy can be geared to the patient(personalized medicine, patient stratification), particularly also inthe set-up to prevent escape as described herein before. It is knownthat the HLA restriction patterns of the Asian population and the blackpopulation are different from the Caucasian population. For differentpopulations different MHC-peptide complexes can be targeted.

Although the present specification presents more specific disclosure ontumors, it must be understood that other aberrant cells can also betargeted by the antibodies of the invention. These other aberrant cellsare typically cells that also proliferate without sufficient control.This occurs in autoimmune diseases. It is typical that these cells startto show expression of tumor antigens. In particular MAGE polypeptideshave been identified in rheumatoid arthritis.^([7])

In literature, it is shown that a single nine amino-acid (A.A.) peptidepresent in MAGE-A2, -A3, -A4, -A6, -A10, and -A12 is presented byHLA-A0201 on tumor cells, and can be recognized by cytotoxicT-lymphocytes.^([1]) This nine amino acid residues peptide with sequenceY-L-E-Y-R-Q-V-P-G (SEQ ID NO:3) is almost identical to the HLA-A0201presented MAGE-A1 peptide Y-L-E-Y-R-Q-V-P-D (SEQ ID NO:5), except forthe anchor residue at position 9. Replacement of the anchor residue withValine results in a nine-amino-acid-residue peptide with enhancedbinding capacity to HLA-A0201 molecules.^([1]) Human and mouseT-lymphocytes recognizing the Y-L-E-Y-R-Q-V-P-V (SEQ ID NO:6) peptidepresented by HLA-0201 also recognize the original MAGE-AY-L-E-Y-R-Q-V-P-G (SEQ ID NO:3) and Y-L-E-Y-R-Q-V-P-D (SEQ ID NO:5)peptides presented on tumors of distinct origin. As diverse tumors mayeach express at least one MAGE-A gene, targeting of this so-calledmulti-MAGE-A epitope includes the vast majority of tumors. As anexample, MAGE-A expression in human prostate tumor cell lines and inhuman xenographs was analyzed and shown to be highly diverse, but ineach individual sample tested at least one MAGE-A gene was expressed(Table 2), confirming that targeting this multi-MAGE-A epitope serves asa universal HLA-A0201 restricted target for therapy.

Of course several other multi-MAGE or multi-target epitopes may bedesigned. In principle the invention contemplates combinations oftumor-specific antigen-derived MHC presented epitopes in different HLArestrictions of both MHC-I and MHC-II, targeted by immunoglobulinslinked to a toxic moiety, to induce apoptosis in aberrant cells.Examples of MHC-MAGE peptide combinations that can be targeted byantibodies hereof are peptide IMPKAGLLI (MAGE-A3) (SEQ ID NO:8) andHLA-DP4 or peptide 243-KKLLTQHFVQENYLEY-258 (MAGE-A3) (SEQ ID NO:9) andHLA-DQ6. Other non-limiting examples of tumor-specific complexes of HLAand antigen peptide are: HLA A1-MAGE-A1 peptide EADPTGHSY (SEQ IDNO:10), HLA A3-MAGE-A1 SLFRAVITK (SEQ ID NO:11), HLA A24-MAGE-A1NYKHCFPEI (SEQ ID NO:12), HLA A28-MAGE-A1 EVYDGREHSA (SEQ ID NO:13), HLAB37-MAGE-A1/A2/A3/A6 REPVTKAEML (SEQ ID NO:14), expressed at aberrantcells related to melanoma, breast carcinoma, SCLC, sarcoma, NSCLC, coloncarcinoma (N. Renkvist et al., Cancer Immunol. Immunother. (2001)V50:3-15 (ref. 13)). Further examples are HLA B53-MAGE-A1 DPARYEFLW (SEQID NO:15), HLA Cw2-MAGE-A1 SAFPTTINF (SEQ ID NO:16), HLA Cw3-MAGE-A1SAYGEPRKL (SEQ ID NO:17), HLA Cw16-MAGE-A1 SAYGEPRKL (SEQ ID NO:18), HLAA2-MAGE A2 KMVELVHFL (SEQ ID NO:19), HLA A2-MAGE-A2 YLQLVFGIEV (SEQ IDNO:20), HLA A24-MAGE-A2 EYLQLVFGI (SEQ ID NO:21), HLA-A1-MAGE-A3EADPIGHLY (SEQ ID NO:22), HLA A2-MAGE-A3 FLWGPRALV (SEQ ID NO:23), HLAB44-MAGE-A3 MEVDPIGHLY (SEQ ID NO:24), HLA B52-MAGE-A3 WQYFFPVIF (SEQ IDNO:25), HLA A2-MAGE-A4 GVYDGREHTV (SEQ ID NO:26), HLA A34-MAGE-A6MVKISGGPR (SEQ ID NO:27), HLA A2-MAGE-A10 GLYDGMEHL (SEQ ID NO:28), HLACw7-MAGE-A12 VRIGHLYIL (SEQ ID NO:29), HLA Cw16-BAGE AARAVFLAL (SEQ IDNO:30), expressed by for example melanoma, bladder carcinoma, NSCLC,sarcoma, HLA A2-DAM-6/-10 FLWGPRAYA (SEQ ID NO:31), expressed by forexample skin tumors, lung carcinoma, ovarian carcinoma, mammarycarcinoma, HLA Cw6-GAGE-1/-2/-8 YRPRPRRY (SEQ ID NO:32), HLAA29-GAGE-3/-4/-5/-6/-7B YYWPRPRRY (SEQ ID NO:33), both expressed by forexample melanoma, leukemia cells, bladder carcinoma, HLA B13-NA88-AMTQGQHFLQKV (SEQ ID NO:34), expressed by melanoma, HLA A2-NY-ESO-1SLLMWITQCFL (SEQ ID NO:35), HLA A2-NY-ESO-1a SLLMWITQC (SEQ ID NO:36),HLA A2-NY-ESO-1a QLSLLMWIT (SEQ ID NO:37), HLA A31-NY-ESO-1a ASGPGGGAPR(SEQ ID NO:38), the latter four expressed by for example melanoma,sarcoma, B-lymphomas, prostate carcinoma, ovarian carcinoma, bladdercarcinoma.

The disclosure is further described by the following non-limitingExamples.

Abbreviations Used

A.A., amino acid; Ab, antibody; β2-M, CDR, complementarity determiningregion; CHO, Chinese hamster ovary; CT, cancer testis antigens; CTL,cytotoxic T-lymphocyte; E4orf4, adenovirus early region 4 open readingframe; EBV, Epstein-Barr virus; ELISA, enzyme linked immunosorbentassay; HAMLET, human α-lactalbumin made lethal to tumor cells; HEK,human embryonic kidney; HLA, human leukocyte antigen; Ig,immunoglobulin; i.v., intravenously; kDa, kilo Dalton; MAGE,melanoma-associated antigen; Mda-7, melanoma differentiation-associatedgene-7; MHC, major histocompatibility complex; MHC-p, MHC-peptide; NS1,parvovirus-H1-derived non-structural protein 1; PBSM, PBS containing 2%non-fat dry milk; TCR, T-cell receptor; VH, Vh or V_(H), amino-acidsequence of an immunoglobulin variable heavy domain; Vl, amino-acidsequence of an immunoglobulin variable light domain; TRAIL, tumornecrosis factor-related apoptosis-inducing ligand.

EXAMPLES Example 1

Non-exhaustive examples of immunoglobulins hereof comprising at least animmunoglobulin variable region that specifically binds to an MHC-peptidecomplex preferentially associated with aberrant cells or to an aberrantcell surface marker preferentially associated with aberrant cells, withdomain topologies as outlined for example in FIG. 5B, are:

Antibodies hereof comprising immunoglobulin variable regions thatspecifically bind to:

-   -   (a) a complex comprising a T-cell epitope selected from        146-KLQCVDLHV-154 (SEQ ID NO:74), 141-FLTPKKLQCV-150 (SEQ ID        NO:75), 154-VISNDVCAQV-163 (SEQ ID NO:76), 154-YISNDVCAQV-163        (SEQ ID NO:77) of PSA, presented by HLA-A2 and/or        162-QVHPQKVTK-170 (SEQ ID NO:78) of PSA, presented by HLA-A3,        and/or 152-CYASGWGSI-160 (SEQ ID NO:79), 248-HYRKWIKDTI-257 (SEQ        ID NO:80) of PSA, presented by HLA-A24, and/or 4-LLHETDSAV-12        (SEQ ID NO:81), 711-ALFDIESKV-719 (SEQ ID NO:82),        27-VLAGGFFLL-35 (SEQ ID NO:83) of PSMA, presented by HLA-A2,        and/or 178-NYARTEDFF-186 (SEQ ID NO:84), 227-LYSDPADYF-235 (SEQ        ID NO:85), 624-TYSVSFDSL-632 (SEQ ID NO:86) of PSMA, presented        by HLA-A24, and/or 299-ALDVYNGLL-307 (SEQ ID NO:87) of PAP,        presented by HLA-A2 and/or 213-LYCESVHNF-221 (SEQ ID NO:88) of        PAP, presented by HLA-A24 and/or 199-GQDLFGIWSKVYDPL-213 (SEQ ID        NO:89), 228-TEDTMTKLRELSELS-242 (SEQ ID NO:90) of PAP, presented        by MHC-2 and/or 14-ALQPGTALL-22 (SEQ ID NO:91),        105-AILALLPAL-113 (SEQ ID NO:92), 7-ALLMAGLAL-15 (SEQ ID NO:93),        21-LLCYSCKAQV-30 (SEQ ID NO:94) of PSCA, presented by HLA-A2        and/or 155-LLANGRMPTVLQCVN-169 (SEQ ID NO:95) of Kallikrein 4,        presented by DRB1*0404 and/or 160-RMPTVLQCVNVSVVS-174 (SEQ ID        NO:96) of Kallikrein 4, presented by DRB1*0701 and/or        125-SVSESDTIRSISIAS-139 (SEQ ID NO:97) of Kallikrein 4,        presented by DPB1*0401, for the treatment of prostate cancer;    -   (b) the HLA B8 restricted epitope from EBV nuclear antigen 3,        FLRGRAYGL (SEQ ID NO:98), complexed with MHC I, for the        clearance of EBV infected cells;    -   (c) the MAGE-A peptide YLEYRQVPG (SEQ ID NO:3) presented by MHC        1 HLA-A0201, for treatment of cancers accompanied by tumor cells        expressing these MHC-peptide complexes (see Table 1);    -   (d) the MAGE-A peptide EGDCAPEEK (SEQ ID NO:4) presented by        MHC-1 HLA-CW7, for treatment of cancers accompanied by tumor        cells expressing these MHC-peptide complexes (see Table 1);    -   (e) complexes of HLA-A2 and HLA-A2 restricted CD8⁺ T-cell        epitopes, e.g., nonamer peptides FLFLLFFWL (SEQ ID NO:99) (from        prostatic acid phosphatase (PAP, also prostatic-specific acid        phosphatase (PSAP))), TLMSAMTNL (SEQ ID NO:100) (from PAP),        ALDVYNGLL (SEQ ID NO:101) (from PAP), human HLA-A2.1-restricted        CTL epitope ILLWQPIPV (SEQ ID NO:102) (from PAP-3),        six-transmembrane epithelial antigen of prostate (STEAP), or        complexes of HLA-A2.1 and HLA-A2.1-restricted CTL epitope        LLLGTIHAL (SEQ ID NO:103) (from STEAP-3), epitopes from mucin        (MUC-1 and MUC-2), MUC-1-32mer        (CHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPA (SEQ ID NO:104)), epitopes        from Globo H, Lewisy, Tn(c), TF(c) clusters, GM2,        prostate-specific membrane antigen (PSMA), kallikrein 4,        prostein, or complexes of HLA-A2.1 and HLA-A2.1-restricted        epitopes from BA46, PTH-rP, HER-2/neu, hTERT, and MAGE-A8, for        the treatment of prostate cancer;    -   (f) an aberrant cell-specific epitope in aberrant cell-specific        altered MUC-1 complexed with MHC, or to an aberrant        cell-specific epitope in aberrant cell-specific altered MUC-1        for, the targeting of aberrant cells in for example breast        cancer or for the treatment of colorectal cancer;    -   (g) an aberrant cell-specific epitope of the aberrant        cell-specific epidermal growth factor receptor mutant form vIII        complexed with MHC, or to an aberrant cell-specific epitope of        the epidermal growth factor receptor mutant form vIII, for the        treatment of the brain neoplasm glioblastoma multiforme;    -   (h) the complex of MHC with T-cell epitope peptide 369-376 from        human Her-2/neu, for the treatment of malignancies related to        Her-2 and/or Her-1 over-expression;    -   (i) an epitope of the aberrant cell-specific surface marker CD44        splice variants known as CD44-v6, CD44-v9, CD44-v10, complexed        with MHC, or to an aberrant cell-specific epitope of an aberrant        cell-specific CD44 splice variant, for the treatment of multiple        myeloma;

Target binding sites suitable for specific and selective targeting ofinfected aberrant cells by antibodies hereof are pathogen-derivedantigen peptides complexed with MHC molecules. Examples of T-cellepitopes of the E6 and E7 protein of human papilloma virus, complexedwith indicated HLA molecules, are provided below. Any combination of anHLA molecule complexed with a pathogen-derived T-cell epitope provides aspecific target on infected aberrant cells for antibodies hereof. Anexample of an infected aberrant cell is a keratinocyte in the cervixinfected by human papilloma virus (HPV), presenting T-cell epitopesderived from for example E6 or E7 protein, in the context of MHC.Examples of suitable target HPV 16 E6 T-cell epitopes are peptidesFQDPQERPR (SEQ ID NO:39), TTLEQQYNK (SEQ ID NO:40), ISEYRHYCYS (SEQ IDNO:41) and GTTLEQQYNK (SEQ ID NO:42) binding to HLA A1, KISEYRHYC (SEQID NO:43) and YCYSIYGTTL (SEQ ID NO:44) binding to HLA A2, LLRREVYDF(SEQ ID NO:45) and IVYRDGNPY (SEQ ID NO:46) binding to HLA A3, TTLEQQYNK(SEQ ID NO:47) binding to HLA All, CYSLYGTTL (SEQ ID NO:48), KLPQLCTEL(SEQ ID NO:49), HYCYSLYGT (SEQ ID NO:50), LYGTTLEQQY (SEQ ID NO:51),EVYDFAFRDL (SEQ ID NO:52) and VYDFAFRDLC (SEQ ID NO:53) binding to HLAA24, 29-TIHDIILECV-38 (SEQ ID NO:54) binding to HLA A*0201. Equallysuitable are HPV 16 E7 T-cell epitopes such as 86-TLGIVCPI-93 (SEQ IDNO:55), 82-LLMGTLGIV-90 (SEQ ID NO:56), 85-GTLGIVCPI-93 (SEQ ID NO:57)and 86-TLGIVCPIC-94 (SEQ ID NO:58) binding to HLA A*0201, HPV 18 E6T-cell epitopes and HPV 18 E7 T-cell epitopes, binding to HLA A1, A2,A3, All or A24. Yet additional examples of T-cell epitopes related toHPV infected cells are HPV E7-derived peptides 1-MHGDTPTLHEYD-12 (SEQ IDNO:59), 48-DRAHYNIVTFCCKCD-62 (SEQ ID NO:60) and 62-DSTLRLCVQSTHVD-75(SEQ ID NO:61) binding to HLA DR, 7-TLHEYMLDL-15 (SEQ ID NO:62),11-YMLDLQPETT-20 (SEQ ID NO:63), 11-YMLDLQPET-19 (SEQ ID NO:64) and12-MLDLQPETT-20 (SEQ ID NO:65) binding to HLA A*201, 16-QPETTDLYCY-25(SEQ ID NO:66), 44-QAEPDRAHY-52 (SEQ ID NO:67) and 46-EPDRAHYNIV-55 (SEQID NO:68) binding to HLA B18, 35-EDEIDGPAGQAEPDRA-50 (SEQ ID NO:69)binding to HLA DQ2, 43-GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR-77 (SEQ IDNO:70) binding to HLA DR3, 50-AHYNIVTFCCKCD-62 (SEQ ID NO:71) binding toHLA DR15, 58-CCKCDSTLRLC-68 (SEQ ID NO:72) binding to HLA DR17 and61-CDSTLRLCVQSTHVDIRTLE-80 (SEQ ID NO:73) binding to HLA-DRB1*0901.

A good source for selecting binding sites suitable for specific andselective targeting of aberrant cells by antibodies hereof, is thePeptide Database listing T-cell defined tumor antigens and the HLAsbinding the T-cell epitopes^([9-12]) (on the WorldWideWeb atcancerimmunity.org/peptidedatabase/Tcellepitopes.htm). The databaseprovides combinations of antigen peptides complexed with MHC moleculescomprising the indicated class of HLA, unique to tumor cells orover-expressed by tumor cells.

Example 2 Selection of Human Antibody Fragments Specific forHLA-A0201/Multi-MAGE-A

To obtain human antibody fragments comprising immunoglobulin variableregions specific for the HLA-A0201 presented multi-MAGE-A epitopeY-L-E-Y-R-Q-V-P-V (SEQ ID NO:6) and FLWGPRALV (SEQ ID NO:23) a Human Fabphage display library was constructed according to the procedurepreviously described by de Haard et al.^([2]) and used for selections 1)essentially as described by Chames et al. using biotinylated MHC/pcomplexes,^([3]) or 2) on cells expressing the relevant antigen.

2.1: Selection of Human Antibody Fragments Specific forHLA-A0201/YLEYRQVPV (SEQ ID NO:6) Using Biotinylated MHC-PeptideComplexes:

Human Fab phages (10¹³ colony forming units) were first pre-incubatedfor one hour at room temperature in PBS containing 2% non-fat dry milk(PBSM). In parallel, 200 μl Streptavidin-coated beads (Dynal™) wereequilibrated for one hour in PBSM. For subsequent rounds, 100 μl beadswere used. To deplete for pan-MHC binders, each selection round, 200 nMof biotinylated MHC class I-peptide (MHC-p) complexes containing anirrelevant peptide (Sanquin, the Netherlands) were added to the phagesand incubated for 30 minutes under rotation. Equilibrated beads wereadded, and the mixture was incubated for 15 minutes under rotation.Beads were drawn to the side of the tube using magnetic force. To thedepleted phage fraction, subsequently decreasing amounts of biotinylatedMHC-p complexes (200 nM for the first round, and 20 nM for the secondand third round) were added and incubated for one hour at roomtemperature, with continuous rotation. Simultaneously, a pan-MHC class Ibinding soluble Fab (D3) was added to the phage-MHC-p complex mixture(50, 10, and 5 μg for rounds 1-3, respectively). Equilibratedstreptavidin-coated beads were added, and the mixture was incubated for15 minutes under rotation. Phages were selected by magnetic force.Non-bound phages were removed by five washing steps with PBSM, fivesteps with PBS containing 0.1% TWEEN®, and five steps with PBS. Phageswere eluted from the beads by ten minutes incubation with 500 μl freshlyprepared tri-ethylamine (100 mM). The pH of the solution was neutralizedby the addition of 500 μl 1 M Tris (pH 7.5). The eluted phages wereincubated with logarithmic growing E. Coli TG1 cells (OD_(600nm) of 0.5)for 30 minutes at 37° C. Bacteria were grown overnight on 2×TYAG plates.Next day, colonies were harvested, and a 10 μl inoculum was used in 50ml 2×TYAG. Cells were grown until an OD_(600nm) of 0.5, and 5 ml of thissuspension was infected with M13k07 helper phage (5×10¹¹ colony formingunits). After 30 minutes incubation at 37° C., the cells werecentrifuged, resuspended in 25 ml 2×TYAK, and grown overnight at 30° C.Phages were collected from the culture supernatant as describedpreviously, and were used for the next round panning. After threeselection rounds a 261-fold enrichment was obtained, and 46 out of 282analyzed clones were shown to be specific for the HLA-A2-multi-MAGE-Acomplex (FIG. 1). ELISA using the HLA-A0201/multi-MAGE-A complexes aswell as HLA-A0201 complexes with a peptide derived from JC virus wasused to determine the specificity of the selected Fab.

2.2: Selection of Human Fab Specific for HLA-A0201/FLWGPRALV (SEQ IDNO:23) Using Cells.

Selections of Fab-phages specifically binding to HLA-A0201/FLWGPRALV(SEQ ID NO:23) were performed using mouse CMT64 lung tumor cells. Toobtain CMT64 cells stably expressing HLA-A0201/FLWGPRALV (SEQ ID NO:23)(A2/FLW) complexes, the CMT64 cells were retroviral infected with avector encoding a single chain peptide-β2M-HLA-A0201 heavy chainconstruct (SEQ ID NO:2). Human Fab phages (10¹³ colony forming units)were first pre-incubated for one hour at room temperature in PBScontaining 2% FCS (PBSF). In parallel, 1.0×10⁶ CMT64-A2/FLW cells wereequilibrated for one hour in PBSF. The phages were first incubated forone hour with 10×10⁶ CMT 64 cells expressing HLA-A0210/YLEYRQVPG (SEQ IDNO:3) to deplete non-specifically binding phages. The non-bound fractionwas then incubated (one hour at 4° C.) with HLA-A0201/FLWGPRALV (SEQ IDNO:23) expressing CMT64 cells. After extensive washing, bound phageswere eluted by adding 500 μl freshly prepared tri-ethylamine (100 mM).The pH of the solution was neutralized by the addition of 500 μl 1 MTris (pH 7.5). The eluted phages were incubated with logarithmic growingE. Coli TG1 cells (OD_(600nm) of 0.5) for 30 minutes at 37° C. Bacteriawere grown overnight on 2×TYAG plates. Next day, colonies wereharvested. After four rounds of selection, individual clones wereselected and tested for specificity of binding.

2.3: Human Fab Specific for HLA-A0201/Multi-MAGE-A Epitopes BindAntigen-Positive Cells.

Multi-MAGE-A; Y-L-E-Y-R-Q-V-P-V

Fab phages were analyzed for their capacity to bind HLA-A0201-positiveEBV-transformed B-LCL loaded with the multi-MAGE-A peptideY-L-E-Y-R-Q-V-P-V (SEQ ID NO:6). The B-LCL line BSM (0.5×10⁶) was loadedwith multi-MAGE-A peptide (10 μg in 100 μl PBS) for 30 minutes at 37°C., followed by incubation with the Fab phages AH5, CB1, CG1, BD5 andBC7 and analyzed by flow-cytometry. As shown in FIG. 2, Fab AH5, CB1 andCG1, specifically bound to the peptide loaded cells only, whereas FabBD5 and BC7 displayed non-specific binding to BSM that was not loadedwith the multi-MAGE-A peptide. No binding was observed by AH5, CB1 andCG1 to non-peptide loaded cells.

Phages presenting AH5, CB1 and CG1, as well as the HLA-A0101/MAGE-A1-specific Fab phage G8 (4) were then used to stain tumor cell lines ofdistinct histologic origin. To this end prostate cancer cells (LNCaP),multiple myeloma cells (MDN), melanoma cells (MZ2-MEL43 and G43), andbreast cancer cells (MDA-MB157) were stained and analyzed by flowcytometry (FIG. 3). The Fab AH5 specifically bound multiple myelomacells MDN, and not the HLA-A0201-negative melanoma and breast cancercells. Both CB1 and CG1 displayed non-specific binding on the melanomacell line G43. The positive control Fab G8 demonstrated binding to allcell lines tested.

Multi-MAGE-A: F-L-W-G-P-R-A-L-V (SEQ ID NO:23)

To determine the cell-binding capacity of the HLA-A0201/FLWGPRALV (SEQID NO:23) selected Fab clone F9 soluble Fab fragments were made byinduction of TG-1 bacteria. TG-1 containing pCes-F9 were grown untilOD=0.8 and Fab production was induced by addition of 1 mM IPTG. After 13hours induction the bacterial periplasmic fraction was isolated anddialyzed overnight. Next day soluble Fab F9 fragments were purified byIMAC.

Purified Fab F9 was added to 0.5×10⁶ CMT 64 cells expressing eitherHLA-A0210/YLEYRQVPG (SEQ ID NO:3), HLA-A0201/FLWGPRALV (SEQ ID NO:23),or CMT 64 cells that do not express human HLA. As shown in FIG. 6 theFab clone F9 specifically binds HLA-A0201/FLWGPRALV (SEQ ID NO:23)expressing CMT64 cells and not CMT 64 cells that do not express humanHLA or that do express the irrelevant HLA-A0201/YLEYRQVPG (SEQ ID NO:3)molecules.

2.4: Fab AH5 Binds HLA-A0201/Multi-MAGE-A Complexes Only.

ELISA using multiple peptide/MHC complexes then confirmed thespecificity of Fab-AH5. To this end HLA-A0201 complexes presentingpeptides multi-MAGE-A, gp100, JCV and MAGE-C2, as well as aHLA-A1/MAGE-A1 complex were immobilized on 96-well plates and incubatedwith phages displaying Fab AH5 and control Fab G8. As shown in FIG. 4,AH5 only binds HLA-A0201/multi-MAGE-A and not the irrelevant complexesHLA-A0201/gp100, HLA-A0201/MAGE-C2, HLA-A0201/JCV and HLA-A0101/MAGE-A1.The positive control Fab G8 only binds to its relevant targetHLA-A0101/MAGE-A1.

The nucleic acids encoding for the HLA-A0201-multi-MAGE-A complexbinding Fab AH5 will be combined with nucleic acids encoding for humanantibody Ch2-Ch3 domains, providing nucleic acid molecules encoding fora human antibody light chain encompassing the selected Cl-Vl encodingnucleic acids and encoding for a human antibody heavy chain encompassingthe selected Ch-Vh encoding nucleic acids. These nucleic acid moleculesencoding the desired immunoglobulin will be introduced, via a plasmid orvia an expression vector, into a eukaryotic host cell such as a CHOcell. After expression of the immunoglobulin, it will be isolated fromthe cell culture and purified. Then, a selected toxic moiety will belinked to the immunoglobulin, for example using Universal Linkage Systemlinker chemistry.

Example 3 Cell Binding and Internalization of an Immunoglobulin Providedwith a Toxic Moiety

Binding capacity of an antibody hereof is analyzed by flow-cytometry.For example, an antibody comprising immunoglobulin variable regionsspecific for complexes of HLA-A0201 and the multi-MAGE-A peptide isanalyzed. HLA-A0201/multi-MAGE-A-positive tumor cells (Daju, MDN and mel624) and HLA-A0201/multi-MAGE-A-negative cells (BSM, G43 and 293) areincubated on ice with purified antibody and detected by addition offluorescently labeled antibodies. Cells bound by the antibody arequantified and visualized by flow-cytometry. Internalization of antibodyis analyzed by confocal microscopy. To this end, cells are incubatedwith the antibody, kept on ice for 30 minutes to allow binding but nointernalization. Next, fluorescently labeled antibodies specific for theantibody are added. To induce internalization cells are transferred to37° C. and fixed with 1% PFA after 5, 10 and 15 minutes.

Example 4 Apoptosis Induction by Antibodies Hereof in Diverse TumorCells

4.1: Killing of Diverse Tumor Cells by Immunoglobulin Provided with aToxic Moiety

Antibodies hereof are analyzed for their capacity to induce apoptosis byincubation with diverse tumor cells, known to express the antigenscomprising the binding sites for the immunoglobulin variable regions.For example, an antibody comprising immunoglobulin variable region VHspecific for complexes of HLA-A0201 and the multi-MAGE-A peptide,AH5-BTX, is coupled to a synthetic HPMA polymer containing the BTXpeptide and Doxorubicin (as we described in WO2009131435, the contentsof which are incorporated herein by this reference) and analyzed. Tothis end, antibodies hereof coupled to doxorubicin are analyzed fortheir capacity to induce apoptosis by incubation with diverse tumorcells known to express both HLA-A0201 and MAGE-A genes. The cell-linesDaju, Mel 624 (melanoma), PC346C (prostate cancer), and MDN (multiplemyeloma) as well as MAGE-A-negative cells (911 and HEK293T) areincubated with different concentrations of the antibodies hereof (inDMEM medium, supplemented with pen/strep, Glutamine and non-essentialamino acids). Several hours later, cells are visually inspected forclassical signs of apoptosis such as detachment of the cells from tissueculture plates and membrane blebbing. In addition, cells are stained foractive caspase-3 to demonstrate apoptosis. It is excepted that theantibodies hereof induce apoptosis in the Daju Mel 624, PC346C and MDNcells. Cells that are not treated with the antibodies hereof are notaffected, as well as cells that do not express HLA-A0201 (HEK293T) andMAGE-A genes (911 and HEK293T).

Another antibody, comprising Vh and VI domains (scFv) with specificityfor complexes of HLA-A01, presenting a MAGE-A1 peptide was alsoanalyzed. The scFv-BTX construct was coupled to the HPMA polymercontaining doxorubicin and incubated with MAGE-A1-positive andMAGE-A1-negative cells. Apoptosis is shown by staining for activecaspase-3.

4.2: Detection of Active Caspase-3.

A classical intra-cellular hallmark for apoptosis is the presence ofactive caspase-3. To determine whether or not the antibodies hereofinduce active caspase-3, Daju, Me1624 and MDN cells are incubated withvarious concentrations of antibodies hereof. After four and 13 hoursFAM-DEVD-FMK, a fluorescently caspase-3/7 inhibitor, is added andpositively stained cells are visualized by fluorescent microscopy andflow-cytometry. Caspase-3 activity is shown in antigen-positive cellsand not in antigen-negative cells, with the (fragment of the) antigenproviding the specific target-binding site for the antibodies hereof.

4.3: Treatment of Tumor Bearing Mice with Immunoglobulins Provided witha Toxic Moiety.

Nude mice (NOD-scid, eight per group) with a palpable subcutaneoustransplantable human tumor (Daju or MDN) are injected with differentdoses of immunoglobulins provided with a toxic moiety. As a control miceare treated with standard chemotherapy or receive an injection with PBS.Mice receiving an optimal dose of the immunoglobulins provided with atoxic moiety survive significantly longer that those mice receivingchemotherapy or PBS, when the aberrant cells expose the target bindingsites for the antibodies hereof.

Example 5 5.1: Selection of Llama VHH with Specificity forHLA-A0201/FLWGPRALV (SEQ ID NO:23) and HLA-A0201/YLEYRQVPG (SEQ ID NO:3)

Selection of Llama VHH fragments with specificity forHLA-A0201/FLWGPRALV (SEQ ID NO:23) (A2/FLW) and HLA-A0201/YLEYRQVPG (SEQID NO:3) (A2/YLE) were performed on CMT64 cells stably expressing theseHLA/peptide complexes. Llama VHH phages (10¹¹ colony forming units) werefirst pre-incubated for one hour at room temperature in PBS containing2% FCS (PBSF). In parallel, 1.0×10⁶ CMT64-A2/FLW and 1.0×10⁶ CMT64A2/YLE cells were equilibrated for one hour in PBSF. To deplete fornon-specific binding phages 10×10⁶ CMT 64 cells expressing either A2/FLWor A2/YLE were incubated for one hour with the llama VHH. The non-boundfractions were then incubated (one hour at 4° C.) with A2/FLW or A2/YLEexpressing CMT64 cells. After extensive washing, bound phages wereeluted by adding 500 μl freshly prepared tri-ethylamine (100 mM). The pHof the solution was neutralized by the addition of 500 μl 1 M Tris (pH7.5). The eluted phages were incubated with logarithmic growing E. ColiTG1 cells (OD_(600nm) of 0.5) for 30 minutes at 37° C. Bacteria weregrown overnight on 2×TYAG plates. Next day, colonies were harvested.After four rounds of selection individual clones were selected andtested for specificity of binding.

5.2: Llama VHH Specific for HLA-A0201/Multi-MAGE-A Epitopes BindAntigen-Positive Cells

To determine the cell-binding capacity of the A2/FLW and A2/YLE selectedVHH soluble VHH fragments were made by induction of TG-1 bacteria. TG-1containing pHen-VHH were grown until OD=0.8 and Fab production wasinduced by addition of 1 mM IPTG. After 13 hours induction, thebacterial periplasmic fraction was isolated and dialyzed overnight. Nextday, soluble VHH fragments were purified by IMAC.

CMT 64 cells (0.5×10⁶) expressing either HLA-A0210/YLEYRQVPG (SEQ IDNO:3), HLA-A0201/FLWGPRALV (SEQ ID NO:23), or CMT 64 cells that do notexpress human HLA were incubated with purified VHH fragments for onehour at 4° C. As shown in FIG. 7, the A2/FLW-specific VHH bindHLA-A0201/FLWGPRALV (SEQ ID NO:23) expressing CMT64 cells and not CMT 64cells that do not express human HLA or that do express the irrelevantHLA-A0201/YLEYRQVPG (SEQ ID NO:3) molecules. The A2/YLE-specific VHHonly bind HLA-A2/YLEYRQVPG (SEQ ID NO:3) expressing CMT64 cells and notA2/FLW-positive CMT64 cells and CMT64 cells that do not express humanHLA.

REFERENCES The Contents of Each of which are Incorporated Herein by thisReference

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TABLE 1 Examples of the frequency of MAGE-A expression by human cancers.Frequency of expression (%) Cancer MAGE-A1 MAGE-A2 MAGE-A3 MAGE-A4MAGE-A6 MAGE-A10 MAGE-A11 Melanoma 16 E 36 E 64 E 74 Head and neck 25 4233 8 N N N Bladder 21 30 35 33 15 N 9 Breast 6 19 10 13 5 N N ColorectalN 5 5 N 5 N N Lung 21 30 46 11 8 N N Gastric 30 22 57 N N N N Ovarian 5532 20 E 20 N N Osteosarcoma 62 75 62 12 62 N N hepatocarcinoma 68 30 68N 30 30 30 Renal cell 22 16 76 30 N N N carcinoma E, expressed but thefrequency is not known; N, expression by tumors has never been observed

TABLE 2 MAGE-A expression in human prostate cancer cell lines andprostate cancer xenografts. Cell line/ MAGE- Xenograft A1 A2 A3 A4 A5 A6A7 A8 A9 A10 A11 A12 LNCaP + ++ ++ ++ + PC346C + ++ ++ + ++ + + ++OVCAR + + + + JON ++ ++ ++ + + PNT 2 C2 + + + + + SD48 + + + +PC-3 + + + PC 374 + PC 346p + ++ ++ ++ + ++ + PC 82 + + PC 133 ++ + + PC135 + PC 295 + PC 324 + + + PC 310 + ++ + ++ + PC 339 ++ ++ + ++ + + +Expression of the MAGE-A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11 andA12 genes in diverse prostate tumor cell lines and prostate xenograftswas analyzed by RT-PCR. Shown are expression levels in individualsamples tested. Blank = no expression, + = low expression, ++ = highexpression. All cell lines/xenografts express at least one MAGE-A gene.

SEQUENCE IDENTIFIERS SEQ ID NO: l. Amino acid sequence Vh AH5QLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTV SSGSTSGSSEQ ID NO: 2, single chain HLA-A0201/FLWGPRALV construct.MAVMAPRTLVLLLSGALALTQTWAFLWGPRALVGGGGSGGGGSGGGGSGGGSGIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSESHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDSPKAHVTHHPRSKGEVTLRCWALGFYPADITLTWQLNGEELTQDMELVETRPAGDGTFQKWASVVVPLGKEQNYTCRVYHEGLPEPLTLRWEPPPSTDSYMVIVAVLGVLGAMAIIGAVVAFVMKRRRNTGGGDYALAPGSQSSEMSLRDCKASEQ ID NO: 3. Amino acid sequence MHC-1 HLA-A0201 presentable peptide in MAGE-AYLEYRQVPGSEQ ID NO: 4. Amino acid sequence MHC-1 HLA-CW7 presentable peptide in MAGE-AEGDCAPEEKSEQ ID NO: 5. Amino acid sequence MHC-1 HLA-A0201 presentable peptide in MAGE-A1YLEYRQVPDSEQ ID NO: 6. Amino acid sequence MHC-1 HLA-A0201 presentable peptide in MAGE-A1,with enhanced binding capacity for HLA-A0201 YLEYRQVPVSEQ ID NO: 7. Amino acid sequence Vh binding domain 11HEVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWLSYISSDGSTIYYADSVKGRFTVSRDNAKNSLSLQMNSLRADDTAVYYCAVSPRGYYYYGLDLWGQGTT VTVSSSEQ ID NO: 8, amino acid sequence of MAGE-A3 peptide epitope binding to HLAIMPKAGLLISEQ ID NO: 9, amino acid sequence of MAGE-A3 peptide epitope binding to HLAKKLLTQHFVQENYLEYSEQ ID NO: 10, amino acid sequence of MAGE peptide epitope binding to HLAEADPTGHSYSEQ ID NO: 11, amino acid sequence of MAGE peptide epitope binding to HLASLFRAVITKSEQ ID NO: 12, amino acid sequence of MAGE peptide epitope binding to HLANYKHCFPEISEQ ID NO: 13, amino acid sequence of MAGE peptide epitope binding to HLAEVYDGREHSASEQ ID NO: 14, amino acid sequence of MAGE peptide epitope binding to HLAREPVTKAEMLSEQ ID NO: 15, amino acid sequence of MAGE peptide epitope binding to HLADPARYEFLWSEQ ID NO: 16 amino acid sequence of MAGE peptide epitope binding to HLASAFPTTINFSEQ ID NO: 17, amino acid sequence of MAGE peptide epitope binding to HLASAYGEPRKLSEQ ID NO: 18, amino acid sequence of MAGE peptide epitope binding to HLASAYGEPRKLSEQ ID NO: 19, amino acid sequence of MAGE peptide epitope binding to HLAKMVELVHFLSEQ ID NO: 20, amino acid sequence of MAGE peptide epitope binding to HLAYLQLVFGIEVSEQ ID NO: 21, amino acid sequence of MAGE peptide epitope binding to HLAEYLQLVFGISEQ ID NO: 22, amino acid sequence of MAGE peptide epitope binding to HLAEADPIGHLYSEQ ID NO: 23, amino acid sequence of MAGE peptide epitope binding to HLAFLWGPRALVSEQ ID NO: 24, amino acid sequence of MAGE peptide epitope binding to HLAMEVDPIGHLYSEQ ID NO: 25, amino acid sequence of MAGE peptide epitope binding to HLAWQYFFPVIFSEQ ID NO: 26, amino acid sequence of MAGE peptide epitope binding to HLAGVYDGREHTVSEQ ID NO: 27, amino acid sequence of MAGE peptide epitope binding to HLAMVKISGGPRSEQ ID NO: 28, amino acid sequence of MAGE peptide epitope binding to HLAGLYDGMEHLSEQ ID NO: 29, amino acid sequence of MAGE peptide epitope binding to HLAVRIGHLYILSEQ ID NO: 30, amino acid sequence of BAGE peptide epitope binding to HLAAARAVFLALSEQ ID NO: 31, amino acid sequence of DAM-6 and DAM-10 peptide epitope binding to HLAFLWGPRAYASEQ ID NO: 32, amino acid sequence of GAGE-1/-2/-8 peptide epitope binding to HLAYRPRPRRYSEQ ID NO: 33, amino acid sequence of GAGE-3/-4/-5/-6/-7B peptide epitope binding to HLAYYWPRPRRYSEQ ID NO: 34, amino acid sequence of NA88-A peptide epitope binding to HLAMTQGQHFLQKVSEQ ID NO: 35, amino acid sequence of NY-ESO-1 peptide epitope binding to HLASLLMWITQCFLSEQ ID NO: 36, amino acid sequence of NY-ESO-1a peptide epitope binding to HLASLLMWITQCSEQ ID NO: 37, amino acid sequence of NY-ESO-1a peptide epitope binding to HLAQLSLLMWITSEQ ID NO: 38, amino acid sequence of NY-ESO-1a peptide epitope binding to HLAASGPGGGAPR SEQ ID NO: 39, HPV 16 E6 T-cell epitope binding to HLA A1FQDPQERPR SEQ ID NO: 40, HPV 16 E6 T-cell epitope binding to HLA A1TTLEQQYNK SEQ ID NO: 41, HPV 16 E6 T-cell epitope binding to HLA A1ISEYRHYCYS SEQ ID NO: 42, HPV 16 E6 T-cell epitope binding to HLA A1GTTLEQQYNK SEQ ID NO: 43, HPV 16 E6 T-cell epitope binding to HLA A2KISEYRHYC SEQ ID NO: 44, HPV 16 E6 T-cell epitope binding to HLA A2YCYSIYGTTL SEQ ID NO: 45, HPV 16 E6 T-cell epitope binding to HLA A3LLRREVYDF SEQ ID NO: 46, HPV 16 E6 T-cell epitope binding to HLA A3IVYRDGNPY SEQ ID NO: 47, HPV 16 E6 T-cell epitope binding to HLA A11TTLEQQYNK SEQ ID NO: 48, HPV 16 E6 T-cell epitope binding to HLA A24CYSLYGTTL SEQ ID NO: 49, HPV 16 E6 T-cell epitope binding to HLA A24KLPQLCTEL SEQ ID NO: 50, HPV 16 E6 T-cell epitope binding to HLA A24HYCYSLYGT SEQ ID NO: 51, HPV 16 E6 T-cell epitope binding to HLA A24LYGTTLEQQY SEQ ID NO: 52, HPV 16 E6 T-cell epitope binding to HLA A24EVYDFAFRDL SEQ ID NO: 53, HPV 16 E6 T-cell epitope binding to HLA A24VYDFAFRDLC SEQ ID NO: 54, HPV 16 E6 T-cell epitope binding to HLA A*020129-TIHDIILECV-38SEQ ID NO: 55, HPV 16 E7 T-cell epitope binding to HLA A*020186-TLGIVCPI-93SEQ ID NO: 56, HPV 16 E7 T-cell epitope binding to HLA A*020182-LLMGTLGIV-90SEQ ID NO: 57, HPV 16 E7 T-cell epitope binding to HLA A*020185-GTLGIVCPI-93SEQ ID NO: 58, HPV 16 E7 T-cell epitope binding to HLA A*020186-TLGIVCPIC-94 SEQ ID NO: 59, HPV E7 T-cell epitope binding to HLA DR1-MHGDTPTLHEYD-12 SEQ ID NO: 60, HPV E7 T-cell epitope binding to HLA DR48-DRAHYNIVTFCCKCD-62SEQ ID NO: 61, HPV E7 T-cell epitope binding to HLA DR62-DSTLRLCVQSTHVD-75SEQ ID NO: 62, HPV E7 T-cell epitope binding to HLA A*201 7-TLHEYMLDL-15SEQ ID NO: 63, HPV E7 T-cell epitope binding to HLA A*20111-YMLDLQPETT-20SEQ ID NO: 64, HPV E7 T-cell epitope binding to HLA A*20111-YMLDLQPET-19SEQ ID NO: 65, HPV E7 T-cell epitope binding to HLA A*20112-MLDLQPETT-20 SEQ ID NO: 66, HPV E7 T-cell epitope binding to HLA B1816-QPETTDLYCY-25 SEQ ID NO: 67, HPV E7 T-cell epitope binding to HLA B1844-QAEPDRAHY-52 SEQ ID NO: 68, HPV E7 T-cell epitope binding to HLA B1846-EPDRAHYNIV-55 SEQ ID NO: 69, HPV E7 T-cell epitope binding to HLA DQ235-EDEIDGPAGQAEPDRA-50SEQ ID NO: 70, HPV E7 T-cell epitope binding to HLA DR343-GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR-77SEQ ID NO: 71, HPV E7 T-cell epitope binding to HLA DR1550-AHYNIVTFCCKCD-62SEQ ID NO: 72, HPV E7 T-cell epitope binding to HLA DR1758-CCKCDSTLRLC-68SEQ ID NO: 73, HPV E7 T-cell epitope binding to HLA-DRB1*090161-CDSTLRLCVQSTHVDIRTLE-80SEQ ID NO: 74, PSA T-cell epitope binding to HLA-A2 146-KLQCVDLHV-154SEQ ID NO: 75, PSA T-cell epitope binding to HLA-A2 141-FLTPKKLQCV-150SEQ ID NO: 76, PSA T-cell epitope binding to HLA-A2 154-VISNDVCAQV-163SEQ ID NO: 77, PSA T-cell epitope binding to HLA-A2 154-YISNDVCAQV-163SEQ ID NO: 78, PSA T-cell epitope binding to HLA-A3 162-QVHPQKVTK-170SEQ ID NO: 79, PSA T-cell epitope binding to HLA-A24 152-CYASGWGSI-160SEQ ID NO: 80, PSA T-cell epitope binding to HLA-A24 248-HYRKWIKDTI-257SEQ ID NO: 81, PSMA T-cell epitope binding to HLA-A2 4-LLHETDSAV-12SEQ ID NO: 82, PSMA T-cell epitope binding to HLA-A2 711-ALFDIESKV-719SEQ ID NO: 83, PSMA T-cell epitope binding to HLA-A2 27-VLAGGFFLL-35SEQ ID NO: 84, PSMA T-cell epitope binding to HLA-A24 178-NYARTEDFF-186SEQ ID NO: 85, PSMA T-cell epitope binding to HLA-A24 227-LYSDPADYF-235SEQ ID NO: 86, PSMA T-cell epitope binding to HLA-A24 624-TYSVSFDSL-632SEQ ID NO: 87, PAP T-cell epitope binding to HLA-A2 299-ALDVYNGLL-307SEQ ID NO: 88, PAP T-cell epitope binding to HLA-A24 213-LYCESVHNF-221SEQ ID NO: 89, PAP T-cell epitope binding to MHC-2199-GQDLFGIWSKVYDPL-213SEQ ID NO: 90, PAP T-cell epitope binding to MHC-2228-TEDTMTKLRELSELS-242SEQ ID NO: 91, PSCA T-cell epitope binding to HLA-A2 14-ALQPGTALL-22SEQ ID NO: 92, PSCA T-cell epitope binding to HLA-A2 105-AILALLPAL-113SEQ ID NO: 93, PSCA T-cell epitope binding to HLA-A2 7-ALLMAGLAL-15SEQ ID NO: 94, PSCA T-cell epitope binding to HLA-A2 21-LLCYSCKAQV-30SEQ ID NO: 95, Kallikrein 4 T-cell epitope binding to DRB1*0404155-LLANGRMPTVLQCVN-169SEQ ID NO: 96, Kallikrein 4 T-cell epitope binding to DRB1*0701160-RMPTVLQCVNVSVVS-174SEQ ID NO: 97, Kallikrein 4 T-cell epitope binding to DPB1*0401125-SVSESDTIRSISIAS-139SEQ ID NO: 98, EBV nuclear antigen 3 T-cell epitope binding to MHC I HLA B8FLRGRAYGLSEQ ID NO: 99, HLA-A2 restricted CD8⁺ T-cell epitope of PAP binding to HLA-A2FLFLLFFWLSEQ ID NO: 100, HLA-A2 restricted CD8⁺ T-cell epitope of PAP binding to HLA-A2TLMSAMTNLSEQ ID NO: 101, HLA-A2 restricted CD8⁺ T-cell epitope of PAP binding to HLA-A2ALDVYNGLLSEQ ID NO: 102, human HLA-A2.1-restricted CTL epitope of PAP-3 binding to HLA A2.1ILLWQPIPVSEQ ID NO: 103, HLA-A2.1-restricted CTL epitope of STEAP-3 binding to HLA-A2.1LLLGTIHALSEQ ID NO: 104, HLA-A2.1-restricted CTL epitope of MUC-1 and MUC-2 binding toHLA-A2.1 CHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPASEQ ID NO: 105, (GlySerThrSerGlySer)n Ig linkerGSTSGSGKPGSGEGSTKGGFAKTTAPSVYPLAPVLESSGSGSEQID NO: 106, IgG1 Ch1-Ch2 hinge region linker EPKSCDKTHTSEQ ID NO: 107, IgG3 hinge region linker ELKTPLGDTTHTSEQ ID NO: 108, IgG4 hinge region linker ESKYGPP

1. An immunoglobulin provided with a toxic moiety, the immunoglobulincomprising at least an immunoglobulin variable region that specificallybinds to an MHC-peptide complex preferentially associated with aberrantcells.
 2. The immunoglobulin of claim 1, wherein the immunoglobulinvariable region is a Vh or Vhh.
 3. The immunoglobulin of claim 2,wherein the immunoglobulin variable region further comprises a Vl. 4.The immunoglobulin of claim 3, which is a human IgG.
 5. Theimmunoglobulin of claim 1, wherein the MHC-peptide complex is specificfor aberrant cells.
 6. The immunoglobulin of claim 1, wherein the toxicmoiety is chemically linked to the immunoglobulin.
 7. The immunoglobulinof claim 1, wherein the toxic moiety is a fusion protein, fused to theimmunoglobulin at the DNA level.
 8. A pharmaceutical compositioncomprising the immunoglobulin of claim 1, and suitable diluents and/orexcipients.
 9. A method of treatment of a host suffering from a diseaseassociated with aberrant cells, comprising: utilizing the immunoglobulinof claim 1 to treat the host.
 10. The method according to claim 9,wherein the toxic moiety is internalized into an aberrant cell.
 11. Themethod according to claim 9 to treat cancer.
 12. The method according toclaim 10 to treat cancer.
 13. An immunoglobulin provided with a toxicmoiety according to FIG. 5, Panel B.
 14. The immunoglobulin of claim 1,wherein the MHC-peptide complex is specific for aberrant cells, througha peptide derived from MAGE.
 15. The immunoglobulin of claim 14, whereinthe MHC-peptide complex is specific for aberrant cells, through apeptide derived from MAGE-A.
 16. The immunoglobulin of claim 7, whereinthe fusion protein is fused to the immunoglobulin at the DNA levelthrough a linking sequence.
 17. A human IgG immunoglobulin chemicallylinked to a toxic moiety, wherein the human IgG immunoglobulin comprisesat least an immunoglobulin variable region that specifically binds to anMHC-peptide complex preferentially associated with aberrant cells,wherein the MHC-peptide complex is specific for aberrant cells through apeptide derived from MAGE-A, and wherein the toxic moiety is a fusionprotein.